The present application relates to a device for holding, puncturing and manipulating the contents of a vesicular object having a size typically in the neighborhood of 100-300 microns. This device has particular application for manipulation of mammalian embryos at the hatched blastocyst stage of development. This has not been possible with any previous device.
Working with the hatched blastocyst has been difficult because the blastocysts are spherical vesicular structures consisting of a thin envelope of living cells surrounding a relatively large central cavity that is filled with an aqueous fluid. The difficulty, and the failure of the prior art, arises because of the physical characteristics of the envelope of these living cells which may be flimsy but resistant to puncture. For example, holding a hatched blastocyst by vacuum from one side with a conventional glass holding pipette while attempting to puncture it by compression from the opposite side with a conventional glass injection pipette often simply indents the blastocyst surface without puncturing it, or conversely, ends up destroying the integrity of the envelope of living cells altogether. This compression can be severe and potentially damaging to the living cells. With the present device there is no compression at all. A portion of the nearside of the surface of the embryo can be grasped and stretched tight for easy penetration.
Additionally, conventional methods disclosed by the prior art require expensive micromanipulators for positioning of the two pipettes required (one for holding and one for injection) in 3-axes so they are perfectly aligned for puncture of the vesicular object by compression. Because the forces compressing the spherical embryo have to be exactly aligned for penetration, both the holding and injection pipettes, presented from above, must have a 30 degree bend near the tip so they can be parallel to the floor of the dish and to each other. None of this is necessary with the present device.